Roman V. Uzhachenko

Sirt3 Impairment and SOD2 Hyperacetylation in Vascular Oxidative Stress and Hypertension

Rationale: Clinical studies have shown that Sirt3 (Sirtuin 3) expression declines by 40% by 65 years of age paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 because of increased NADH (nicotinamide adenine dinucleotide, reduced form) and acetyl-CoA levels. Sirt3 impairment reduces the activity of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2) because of hyperacetylation. Objective: In this study, we examined whether the loss of Sirt3 activity increases vascular oxidative stress because of SOD2 hyperacetylation and promotes endothelial dysfunction and hypertension. Methods and Results: Hypertension was markedly increased in Sirt3-knockout (Sirt3−/−) and SOD2-depleted (SOD2+/−) mice in response to low dose of angiotensin II (0.3 mg/kg per day) compared with wild-type C57Bl/6J mice. Sirt3 depletion increased SOD2 acetylation, elevated mitochondrial O2· –, and diminished endothelial nitric oxide. Angiotensin II-induced hypertension was associated with Sirt3 S-glutathionylation, acetylation of vascular SOD2, and reduced SOD2 activity. Scavenging of mitochondrial H2O2 in mCAT mice expressing mitochondria-targeted catalase prevented Sirt3 and SOD2 impairment and attenuated hypertension. Treatment of mice after onset of hypertension with a mitochondria-targeted H2O2 scavenger, mitochondria-targeted hydrogen peroxide scavenger ebselen, reduced Sirt3 S-glutathionylation, diminished SOD2 acetylation, and reduced blood pressure in wild-type but not in Sirt3−/− mice, whereas an SOD2 mimetic, (2-[2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino]-2-oxoethyl) triphenylphosphonium (mitoTEMPO), reduced blood pressure and improved vasorelaxation both in Sirt3−/− and wild-type mice. SOD2 acetylation had an inverse correlation with SOD2 activity and a direct correlation with the severity of hypertension. Analysis of human subjects with essential hypertension showed 2.6-fold increase in SOD2 acetylation and 1.4-fold decrease in Sirt3 levels, whereas SOD2 expression was not affected. Conclusions: Our data suggest that diminished Sirt3 expression and redox inactivation of Sirt3 lead to SOD2 inactivation and contributes to the pathogenesis of hypertension.

Fus1/Tusc2 Is a Novel Regulator of Mitochondrial Calcium Handling, Ca2+-Coupled Mitochondrial Processes, and Ca2+-Dependent NFAT and NF-κB Pathways in CD4+ T Cells

AIMS Fus1 has been established as mitochondrial tumor suppressor, immunomodulator, and antioxidant protein, but molecular mechanism of these activities remained to be identified. Based on putative calcium-binding and myristoyl-binding domains that we identified in Fus1, we explored our hypothesis that Fus1 regulates mitochondrial calcium handling and calcium-coupled processes. RESULTS Fus1 loss resulted in reduced rate of mitochondrial calcium uptake in calcium-loaded epithelial cells, splenocytes, and activated CD4(+) T cells. The reduced rate of mitochondrial calcium uptake in Fus1-deficient cells correlated with cytosolic calcium increase and dysregulation of calcium-coupled mitochondrial parameters, such as reactive oxygen species production, ΔμH(+), mitochondrial permeability transition pore opening, and GSH content. Inhibition of calcium efflux via mitochondria, Na(+)/Ca(2+) exchanger significantly improved the mitochondrial calcium uptake in Fus1(-/-) cells. Ex vivo analysis of activated CD4(+) T cells showed Fus1-dependent changes in calcium-regulated processes, such as surface expression of CD4 and PD1/PD-L1, proliferation, and Th polarization. Fus1(-/-) T cells showed increased basal expression of calcium-dependent NF-κB and NFAT targets but were unable to fully activate these pathways after stimulation. INNOVATION Our results establish Fus1 as one of the few identified regulators of mitochondrial calcium handling. Our data support the idea that alterations in mitochondrial calcium dynamics could lead to the disruption of metabolic coupling in mitochondria that, in turn, may result in multiple cellular and systemic abnormalities. CONCLUSION Our findings suggest that Fus1 achieves its protective role in inflammation, autoimmunity, and cancer via the regulation of mitochondrial calcium and calcium-coupled parameters.

Tumour suppressor Fus1 provides a molecular link between inflammatory response and mitochondrial homeostasis.

Fus1, encoded by a 3p21.3 tumour suppressor gene, is down‐regulated, mutated or lost in the majority of inflammatory thoracic malignancies. The mitochondrial localization of Fus1 stimulated us to investigate how Fus1 modulates inflammatory response and mitochondrial function in a mouse model of asbestos‐induced peritoneal inflammation. Asbestos treatment resulted in a decreased Fus1 expression in wild‐type (WT) peritoneal immune cells, suggesting that asbestos exposure may compromise the Fus1‐mediated inflammatory response. Untreated Fus1−/− mice had an ∼eight‐fold higher proportion of peritoneal granulocytes than Fus1+/+ mice, pointing at ongoing chronic inflammation. Fus1−/− mice exhibited a perturbed inflammatory response to asbestos, reflected in decreased immune organ weight and peritoneal fluid protein concentration, along with an increased proportion of peritoneal macrophages. Fus1−/− immune cells showed augmented asbestos‐induced activation of key inflammatory, anti‐oxidant and genotoxic stress response proteins ERK1/2, NFκB, SOD2, γH2AX, etc. Moreover, Fus1−/− mice demonstrated altered dynamics of pro‐ and anti‐inflammatory cytokine expression, such as IFNγ, TNFα, IL‐1A, IL‐1B and IL‐10. ‘Late’ response cytokine Ccl5 was persistently under‐expressed in Fus1−/− immune cells at both basal and asbestos‐activated states. We observed an asbestos‐related difference in the size of CD3+ CD4− CD8− DN T cell subset that was expanded four‐fold in Fus1−/− mice. Finally, we demonstrated Fus1‐dependent basal and asbestos‐induced changes in major mitochondrial parameters (ROS production, mitochondrial potential and UCP2 expression) in Fus1−/− immune cells and in Fus1‐depleted cancer cells, thus supporting our hypothesis that Fus1 establishes its immune‐ and tumour‐suppressive activities via regulation of mitochondrial homeostasis. Copyright

A novel radioprotective function for the mitochondrial tumor suppressor protein Fus1

FUS1/TUSC2 is a mitochondrial tumor suppressor with activity to regulate cellular oxidative stress by maintaining balanced ROS production and mitochondrial homeostasis. Fus1 expression is inhibited by ROS, suggesting that individuals with a high level of ROS may have lower Fus1 in normal tissues and, thus, may be more prone to oxidative stress-induced side effects of cancer treatment, including radiotherapy. As the role of Fus1 in the modulation of cellular radiosensitivity is unknown, we set out to determine molecular mechanisms of Fus1 involvement in the IR response in normal tissues. Mouse whole-body irradiation methodology was employed to determine the role for Fus1 in the radiation response and explore underlying molecular mechanisms. Fus1−/− mice were more susceptible to radiation compared with Fus1+/+ mice, exhibiting increased mortality and accelerated apoptosis of the GI crypt epithelial cells. Following untimely reentrance into the cell cycle, the Fus1−/− GI crypt cells died at accelerated rate via mitotic catastrophe that resulted in diminished and/or delayed crypt regeneration after irradiation. At the molecular level, dysregulated dynamics of activation of main IR response proteins (p53, NFκB, and GSK-3β), as well as key signaling pathways involved in oxidative stress response (SOD2, PRDX1, and cytochrome c), apoptosis (BAX and PARP1), cell cycle (Cyclins B1 and D1), and DNA repair (γH2AX) were found in Fus1−/− cells after irradiation. Increased radiosensitivity of other tissues, such as immune cells and hair follicles was also detected in Fus1−/− mice. Our findings demonstrate a previously unknown radioprotective function of the mitochondrial tumor suppressor Fus1 in normal tissues and suggest new individualized therapeutic approaches based on Fus1 expression.

Bortezomib enhances expression of effector molecules in anti-tumor CD8 + T lymphocytes by promoting Notch-nuclear factor-κB crosstalk

The immunosuppressive tumor microenvironment usurps host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+T-lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Bortezomib also neutralized TGFβ-mediated suppression of IFNγ and granzyme B expression in activated CD8+T-cells. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+T-cells. Moreover, bortezomib promoted CD8+T-cell nuclear factor-κB (NFκB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+T-cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+T-cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on anti-tumor T-cell functions.

Mitochondrial protein Fus1/Tusc2 in premature aging and age-related pathologies: critical roles of calcium and energy homeostasis

Decreased energy production and increased oxidative stress are considered to be major contributors to aging and aging-associated pathologies. The role of mitochondrial calcium homeostasis has also been highlighted as an important factor affecting different pathological conditions. Here, we present evidence that loss of a small mitochondrial protein Fus1 that maintains mitochondrial homeostasis results in premature aging, aging-associated pathologies, and decreased survival. We showed that Fus1KO mice develop multiple early aging signs including lordokyphosis, lack of vigor, inability to accumulate fat, reduced ability to tolerate stress, and premature death. Other prominent pathological changes included low sperm counts, compromised ability of adult stem cells to repopulate tissues, and chronic inflammation. At the molecular level, we demonstrated that mitochondria of Fus1 KO cells have low reserve respiratory capacity (the ability to produce extra energy during sudden energy demanding situations), and show significantly altered dynamics of cellular calcium response. Our recent studies on early hearing and memory loss in Fus1 KO mice combined with the new data presented here suggest that calcium and energy homeostasis controlled by Fus1 may be at the core of its aging-regulating activities. Thus, Fus1 protein and Fus1-dependent pathways and processes may represent new tools and targets for anti-aging strategies.

Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8 + T cell antitumor function

Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.

Abstract 5665: Physical crosstalk between CD8+T and natural killer cells elicits antitumor effector response

The interaction between the innate and adaptive immune components is fundamental for an effective antitumor immunity. Our studies in murine solid tumor models showed that productive antitumor effector response relies on functional crosstalk between innate immune effectors—natural killers (NK), and adaptive immune effectors—cytolytic CD8+T lymphocytes. We found that this lymphocyte cooperativity between CD8+T and NK cells can prevent the development of antigen-escape tumor variants. In this study, we first investigated the role of physical contacts in during the functional crosstalk between CD8+T and NK cells. Since, studying dynamic lymphocyte interactions present extreme challenges, in this study, we engineered a 3D nanofiber matrix to provide lymphocytes a 3D culture environment for a controlled interaction. Confocal imaging showed that CD3/CD28-activated CD8+T cells (CD69+CD25+) formed multiple intercellular contacts with several naive NK cells upon coculture, while naive CD8+T cells made single or no contact with NK cells. In lymphocyte coculture (physical contact possible) we found that activated CD8+T and NK cells cross-regulate each other phenotype wherein NK cells polarize activated CD8+T cells towards “T central memory phenotype” and activated CD8+T lymphocytes induce acquisition of “effector/regulatory phenotype” by naive NK cells. This cross-regulation of lymphocytes disappeared in trans-well system (no physical contact) indicating the necessity of cell-to-cell physical interaction during CD8+T—NK crosstalk. Notably, intercellular physical interaction led to cross regulation of mitoCa2+ oscillations in both activated CD8+T and NK cells. Inhibition of mitochondrial Ca2+ uptake or Na+/Ca2+ exchanger with Ru360 and CGP37157, respectively, mimicked observed alterations in both lymphocytes. Further, NK cells displayed increased oxidative signaling, Tyk2, Jak 1 and 3, Stat2 and Stat6 phosphorylation while inhibiting TCR- and various cytokine receptors-mediated signaling. In turn, NK cells selectively restrain IL-2 signaling in CD8+T cells by dampening activation-induced up-regulation of CD25, Stat5 phosphorylation, IL-2 synthesis and elevation in IL-2 uptake. These data underscore a novel mitochondrial Ca2+ transport-regulated acquisition of activation/regulatory phenotype by NK and CD8+T cells upon their interaction. Understanding the critical factors involved in this NK—CD8+T cells immunological synapse allowing their functional remodeling with the intact tissues in tumor settings will lead to novel strategies for effective cancer immunotherapies, with a potential of relapse-free survival in cancer patients. Citation Format: Roman V. Uzhachenko, Ashutosh Singhal, Shawn J. Goodwin, William H. Hofmeister, Anil Shanker. Physical crosstalk between CD8+T and natural killer cells elicits antitumor effector response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5665. doi:10.1158/1538-7445.AM2017-5665